Fossil records date human use of plants as medicines to at least the middle
Paleolithic age-about 60,000 years ago (Solecki, 1975).
Sequel to an age-long efficacy demonstrated by quite a number of medicinal plants,
almost 80% of the worlds population have incorporated them into their
primary modality of health care (Farnsworth et al.,
1985). Different portions of medicinal plants (root, stem, leaves, seeds
and flowers) contain active constituents (Fett Neto and
DiCosmo, 1992) and have been used as treatment remedies (Pala
et al., 2010).
Chromolaena odorata (Fig. 1) is abundant and widespread in nature. Mabberley (1997) reported that about 165 species of the plant Chromolaena are distributed in tropical and warm temperate regions. It is widely used in Nigeria in traditional medicine practice as an anti-malaria remedy, it can also be traditionally applied to wounds to stop bleeding. In Vietnam and other tropical countries, fresh leaves or decoction of the leaves are used for treatment of leech bites, soft tissue wounds, burns, dentoalveolitis and skin infections (Phan et al., 2001). Studies carried out so far on the aqueous and ethanolic extracts of Chromolaena odorata have provided enough scientific justification for its use in traditional medicine. There are reports from India, the Philippines and Togo showing that Chromolaena odorata leaf extracts exhibited anti-cancer activity on human and mouse cell lines (Vital and Rivera, 2009). Prabhu et al. (2011), isolated essential oil from fresh leaves of Chromolaena odorata which was screened for in-vitro cytotoxicity against human cervical cell line (HeLa), human laryngeal epithelial carcinoma cells (HEp-2) and (NIH-3T3), mouse embryonic fibroblast cancer cell lines; their reports showed that essential oil isolated from fresh leaves of Chromolaena odorata consisting mainly of 5, 6-diethenyl-1- methyl-cyclohexene (44.7%), β-guiane (11.9%), elemol (8.5%) and patchoulene (8.6%), had significant cytotoxic effect with IC50values of 60.3, 67.5 and 72.00 μg mL‾1 towards HeLa, HEp-2 and NIH 3T3 cancer cell lines, respectively.
Earlier reports by Koba et al. (2009), on the cytotoxicity potential of essential oil of Chromolaena odorata on human epidermis cell line Hecate, showed that it had a moderate cytotoxicity potential with IC50 of 700 μL mL‾1 mainly as a result of its neural component.
MATERIALS AND METHODS
Collection and identification of plant material: The leaves of the plant Chromolaena odorata were collected from a local farm in Otolo Nnewi, Nigeria. The plant material was identified by Prof. J. C. Okafor of Tree Crops and Tropical Ecology Center, No. 7 Dona Drive, Off Ihiala Street, Independence Layout, Enugu, Nigeria. Voucher specimen of the plant has been deposited at the Department of Biochemistry, Michael Okpara University of Agriculture, Umudike, Umuahia, Abia State, Nigeria.
Preparation of the plant material: The leaves of the plant were air-dried and ground into fine powder using a blender. Water extraction was done with deionized water (1:10 w/v) by boiling for 5 min after which it was allowed to cool. The ethanolic extract was obtained by cold maceration of the ground samples in 95% ethanol (1:10 w/v) for 24 h. The mixtures were thereafter filtered after each extraction, respectively and freeze dried. The samples were then placed in airtight containers and refrigerated.
Phytochemical screening: The aqueous and ethanolic extracts of Chromolaena odorata were subjected to phytochemical analysis (Harborne, 1984), to assess for the presence of phytochemical constituents. Quantitative analysis of the constituents was done using HP6890 Gas chromatography coupled with flame ionizing detector and powered by HP station Rev. A.09.01  software. Result is as shown in Table 1.
Acute toxicity study: Aqueous and ethanolic extracts of Chromolaena odorata leaves were tested orally for acute toxic effect (after phytochemical screening was done), using the method proposed by Lorke (1983). The method comprises two phases, phase 1 and 2.
In phase 1 nine Wistar albino rats were divided into groups 1, 2 and 3, of 3 animals per group and doses of 10, 100 and 1000 mg kg‾1 body weight of aqueous extracts were administered to the three groups of animals, respectively. The animals were then monitored for signs of toxicity and death within 24 h.
As there was no death recorded, the experiment proceeded to phase 2 where nine Wistar albino rats were also divided into groups 5-7, of three animals per group. The groups consequently received 1600, 2900 and 5000 mg kg‾1, respectively and again monitored for another 24 h for sign of toxicity and death.
The median lethal dose (LD50) was estimated as the geometric mean of the least dose which did not kill any of the animals and that which killed all the animals.
Brine shrimp lethality bioassay: The LC50 of the aqueous extract was determined using the method of Meyer et al. (1982).
Hatching: About 50 mg of Artemisia salina (Leach) eggs, (Interpet. Ltd. England) was added to about 150 mL solution of sea water in a beaker. The mixture was allowed to incubate for 48 h in a warm well ventilated room (22-29°C) under a light source. Larvae (nauplii) were collected with a Pasteur pipette after they had been attracted by the light source.
Brine shrimp assay: Twenty milligrams of the aqueous extract was dissolved in 1 mL of distilled water and dilutions were made to obtain concentrations corresponding to 10, 100 and 1000 ppm in 500 μL of each solution sample. Ten shrimps were transferred into each test-tube and the samples were added. The final volume was made up to 5 mL using sterilized sea water. Each sample was tested in triplicate and incubated for 24 h and finally, the tubes were examined under a magnifying glass and the number of dead shrimps was counted. The mortality percentage of each concentration was calculated as:
This data was represented graphically by plotting log of concentration against percentage death (mortality) from which LC50 for each sample was determine by drawing regression line calculated using regression analysis.
Phytochemical screening of Chromolaena odorata extracts: The relative abundance of the phytochemicals as shown in the aqueous extracts was in the following order: Flavonoids>Saponins>Protease inhibitors>Glycosides>Anthraquinones>Terpenes>Tannin>Saponin >Alkaloids>Allicin.
||Toxicity study of ethanolic and aqueous leaf extracts of Chromolaena odorata against brine-shrimp nauplii
Ethanolic extracts also showed similar pattern but more terpenes and less alkaloid, are reflected in Table 1.
Acute toxicity study of Chromolaena odorata: The result of the acute toxicity study are presented in Table 2.
The result obtained in the study showed that high doses of oral administration of Chromolaena odorata aqueous extract produced symptoms of toxicity and behavioral changes. The animals manifested visible signs of toxicity and recorded mortalities of three animals each for the dose groups of 2900 and 5000 mg kg‾1 body weights, respectively in the phase 2 test. Some of the behavioral changes noticed included; loss of spinal reflex, raising of furs, diarrhea and gait disturbances. However, the phase 1 test did not record any mortality.
The ethanolic extract did not exhibit any visible toxic effect or behavioral changes. Hence, no death was recorded following the oral administration of the ethanolic extract as seen in Table 2. The median lethal doses (LD50) of the aqueous and ethanolic extracts were 2154 and >5000 mg kg‾1, respectively in male Wistar albino rats.
Table 3 shows that the degree of lethality was most with the highest concentration. It also exhibited lethality pattern that was found to be directly proportional to the concentration of the extracts.
Aqueous and ethanolic leaf extracts of Chromolaena odorata were tested for toxic activities using brine shrimp lethality bioassay. The results are presented in Table 3 and Fig. 2. The result from the brine shrimp lethality bioassay show that the amount of Chromolaena odorata (aqueous and ethanolic extracts) needed for 50% cytotoxicity was 324 and 392 μg mL‾1, respectively.
In the acute toxicity study, the LD50 (median lethal dose) was found to be greater than 5000 mg kg‾1 for the ethanolic extract and 2154 mg kg‾1 for the aqueous extract. This difference appears quite significant and probably could be accounted for by the differential amounts of the phytochemical components of the fractional extracts. While the ethanolic extract had greater concentrations of alkaloids, glycosides, sterols, allicin, tannins, terpenes and flavonoids, the aqueous extract had a greater concentration of protease inhibitors and saponins. The greater concentrations of protease inhibitors and saponins that are well known potentially toxic substances may also contribute to this greater toxicity observed with the aqueous extract.
Leeuwen and van Vermeire (2007), suggested that LD50 of chemical substances that are less than 5 mg kg‾1 as highly hazardous, <50 mg kg‾1 as hazardous, <500 mg kg‾1 are moderately toxic and <5000 mg kg‾1 as slightly toxic or having no significant toxic effect.
Based on this report, we suggest the aqueous and ethanol extracts of Chromolaena odorata are not significantly toxic. We therefore conclude that the extracts are relatively safe.
The brine shrimp lethality assay represents a rapid, inexpensive and simple bioassay for testing plant extracts bioactivity which correlates reasonably well with cytotoxic and anti-tumor properties (McLaughlin et al., 1993). In this study, toxicity testing against brine shrimp was conducted using both the aqueous and ethanolic extracts of Chromolaena odorata plant species collected from Nnewi Nigeria. The extracts showed non-significant cytotoxic activities with LC50 values of 324 and 392 μg mL‾1 with aqueous and ethanolic extracts, respectively. Moshi et al. (2010) investigated the brine shrimp toxicity of 30 medicinal plants in Tanzania concluded that extracts with LC50 of <500 μg mL‾1 can be safely said to be practically non-toxic while extracts with LC50 >100 μg mL‾1 but <500 μg mL‾1 can be classified as non-toxic. Progressive percentage of toxic responses was observed in this study, being highest with the highest extract concentration of 1000 μg L‾1. Similar activities had been reported of Vernonia Amygdalina which is of the same family Asteraceae. This has been attributed to the presence of sesquiterpene lactone (Kupchan et al., 1969). Also, Lagnika et al. (2011) reported in their studies that extracts giving LC50 values higher than 100 μg mL‾1 are said to exhibit very low toxicity. All these tend to agree with the relative low toxicity associated with Chromolaena odorata in our study.
The finding in this study is suggestive that the extract may possess some cytotoxic activities at higher dose (1000 μg L‾1) and therefore, may have a potential for therapeutic investigation. We therefore conclude that the extract is relatively safe for consumption.